This program explores innate anti-microbial defense, pro-inflammatory and other signaling mechanisms involving deliberate reactive oxygen species (ROS) production by Nox family NADPH oxidases. Our current focus investigates cellular mechanisms regulating phagocytic and nonphagocytic Nox family NADPH oxidases. The nonphagocytic enzymes (Nox1, Nox4, Duox1, Duox2) are expressed primarily in epithelial cells, notably in mucosal surfaces (lung and gastrointestinal tract), liver, kidney, thyroid and exocrine glands (salivary, mammary), and vascular tissues. ROS produced by these oxidases affect cell migration and differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, and responses to microbial factors, growth factors, hormones, or cytokines. Several non-phagocytic Nox enzymes also serve in host defense and inflammatory processes, as they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by cytokines or by recognition of danger- and pathogen-associated molecular patterns (DAMPs, PAMPs). In 2012, we explored several inflammatory mechanisms in the lung related to excess oxidative stress. We examined airway epithelial responses to histamine, a well-known pro-inflammatory mediator of allergic asthma, and showed that the predominant airway NADPH oxidases (dual oxidases, Duox1 and Duox2) are activated by histamine, leading to release of hydrogen peroxide through stimulation of HR1 histamine receptors, in a calcium-dependent mechanism. We developed several model systems to explore this pathway, including human primary and immortalized airway epithelial cells grown on air-liquid interface culture systems, as well as cell lines that heterologously express HR1, Duox, and their maturation factors (Duox activators). Furthermore, we showed airway epithelial Duox1 expression and hydrogen peroxide release in response to histamine are augmented by prior treatment with Th2 cytokines relevant to allergic disease (i.e., IL-13). These studies provide insight on the causes of excess oxidative stress associated with allergic asthma (increased hydrogen peroxide in asthmatic breath condensates) and suggest new targets for diagnostic and therapeutic interventions for allergic asthma. In other studies on causes of excess inflammation and oxidative stress observed in cystic fibrosis (CF), we are investigating involvement of NADPH oxidases in responses to Pseudomonas aeruginosa, a major pathogen associated with CF disease morbidity and mortality. Our early work identified multiple effects of the redox-active Pseudomonas virulence factor pyocyanin on host cells, including direct inhibition of Duox activity and its microbicidal functions in air-liquid interface cultures. Recently we showed chronic airway epithelial exposure to physiologically relevant pyocyanin levels brought about gene expression profile changes that recapitulate the CF disease phenotype: markers of oxidative stress, wound healing, release of proinflammatory cytokines and chemokines that recruit excess inflammatory cells to the lungs of CF patients. We showed that most Pseudomonas isolates tested from CF patients produce pyocyanin, while others showed airway pyocyanin levels correlate with CF disease severity. Related studies have identified yet another effect of pyocyanin on a novel NADPH oxidase-based host defense mechanism relevant to CF: induction of neutrophil extracellular trap (NET) formation. NETs consist of extruded nuclear DNA webs decorated with microbicidal granule proteins. Although NET formation is considered an important innate immune mechanism beyond intracellular killing, uncontrolled NET release can damage host lung tissues in CF. Here we showed pyocyanin promotes NET formation in a time- and dose-dependent manner and that pyocyanin-derived reactive oxygen species are required for NET release. Inhibitor studies showed involvement of Jun N-terminal Kinase (JNK) and phosphatidylinositol 3-Kinase (PI3K) in NET formation. Pyocyanin-induced NETs also require the NADPH oxidase, because NET release in chronic granulomatous disease neutrophils was greatly reduced;comparison of neutrophils from gp91phox- and p47phox-deficient patients revealed pyocyanin-triggered NET formation is proportional to their residual superoxide production. Our studies identify pyocyanin as the first secreted bacterial toxin that enhances NET formation. Some CF patients benefit from aerosolized DNAse-based therapies, which can dissolve NET structures. The involvement of NADPH oxidase and ROS in pyocyanin-induced NET formation represents a novel mechanism of pyocyanin toxicity that may be targeted therapeutically in CF. In collaborative studies with Dr. F.F. Chu, we are exploring potential roles for Duox2 in murine colitis. Mice deficient in glutathione peroxidases 1 and 2 (GPX) show large variations in the penetrance and severity of colitis in C57BL/6J and 129S1/SvImJ backgrounds. They have mapped a locus contributing to this difference to distal chromosome 2 (∼118-125 mbp) and named it glutathione peroxidase-deficiency-associated colitis 1 (Gdac1). The work was aimed at identify the best gene candidates within the Gdac1 locus contributing to the murine colitis phenotype. Using in silico analysis of 128 genes, based on published colon expression data, the relevance of pathways to colitis, gene mutations, presence of non-synonymous-single-nucleotide polymorphisms (nsSNPs) and whether the nsSNPs are predicted to have an impact on protein function, 42 genes were excluded. Twenty-five genes from the remaining 86 genes were analyzed for expression-quantitative-trait loci, and another 15 genes were excluded. Among the remaining 10 genes, we identified PLA2G4F and DUOX2 as the most likely colitis gene candidates, because GPX metabolizes PLA2G4F and DUOX2 products. Duox2 produces hydrogen peroxide and may gut control microbial populations;DUOX-1 and -2 control microbial populations in mammalian lung and in the gut of several insects and zebrafish. Dysbiosis is a phenotype that differentiates 129S1/SvImJ from C57BL/6J and may be due to strain differences in Doux2 activity currently under investigation. Our studies on Nox4 continue to explore its induction by TGF-beta in processes related to wound healing, fibrosis, cell migration and metastatic activity. We previously demonstrated Nox4 is a TGF-beta/SMAD3-inducible source of reactive oxygen species (ROS) affecting the epithelial-to-mesenchymal transition (EMT) and cell migration in normal and metastatic breast epithelial cells. In efforts aimed at defining factors that contribute to pro-metastatic activity of tumor cells, we investigated involvement of p53 in TGF-beta-regulated Nox4 expression and cell migration. We found that wild-type p53 is a potent suppressor of TGF-beta-induced Nox4 expression, ROS production, and cell migration, whereas expression of tumor-associated mutant p53 proteins (R175H or R280K) causes enhanced Nox4 expression and cell migration in both TGF-beta dependent and independent pathways. Current efforts are focused on identifying signaling intermediates in these pathways that can be targeted to suppress cancer progression and metastasis.

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Kwon, Jaeyul; Wang, Aibing; Burke, Devin J et al. (2016) Peroxiredoxin 6 (Prdx6) supports NADPH oxidase1 (Nox1)-based superoxide generation and cell migration. Free Radic Biol Med 96:99-115
Carré, Aurore; Louzada, Ruy A N; Fortunato, Rodrigo S et al. (2015) When an Intramolecular Disulfide Bridge Governs the Interaction of DUOX2 with Its Partner DUOXA2. Antioxid Redox Signal :
Ueyama, Takehiko; Sakuma, Megumi; Ninoyu, Yuzuru et al. (2015) The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release. J Biol Chem 290:6495-506
Boudreau, H E; Casterline, B W; Burke, D J et al. (2014) Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells. Br J Cancer 110:2569-82
Esworthy, R Steven; Kim, Byung-Wook; Wang, Yufeng et al. (2013) The Gdac1 locus modifies spontaneous and Salmonella-induced colitis in mice deficient in either Gpx2 or Gpx1 gene. Free Radic Biol Med 65:1273-83
Rada, Balazs; Boudreau, Howard E; Park, Jonathan J et al. (2013) Histamine Stimulates Hydrogen Peroxide Production by Bronchial Epithelial Cells via Histamine H1 Receptor and Duox. Am J Respir Cell Mol Biol :
Rada, Balázs; Leto, Thomas L (2013) Pyocyanin effects on respiratory epithelium: relevance in Pseudomonas aeruginosa airway infections. Trends Microbiol 21:73-81
Rada, Balázs; Jendrysik, Meghan A; Pang, Lan et al. (2013) Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase. PLoS One 8:e54205
Waisberg, Michael; Cerqueira, Gustavo C; Yager, Stephanie B et al. (2012) Plasmodium falciparum merozoite surface protein 1 blocks the proinflammatory protein S100P. Proc Natl Acad Sci U S A 109:5429-34
Ambruso, Daniel R; Ellison, Michael A; Thurman, Gail W et al. (2012) Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity. Biochim Biophys Acta 1823:306-15

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